Oxidative Stress, Thiol Reagents, and Membrane Potential Modulate the Mitochondrial Permeability Transition by Affecting Nucleotide Binding to the Adenine Nucleotide Translocase

Abstract

Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 microM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 microM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 microM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.